EP1260592A1 - Biopuce - Google Patents

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Publication number
EP1260592A1
EP1260592A1 EP01112179A EP01112179A EP1260592A1 EP 1260592 A1 EP1260592 A1 EP 1260592A1 EP 01112179 A EP01112179 A EP 01112179A EP 01112179 A EP01112179 A EP 01112179A EP 1260592 A1 EP1260592 A1 EP 1260592A1
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EP
European Patent Office
Prior art keywords
probe
biochip
sequence
escherichia coli
sequences
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EP01112179A
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German (de)
English (en)
Inventor
Horst Donner
Bernd Drescher
Andrea Huber
Jaqueline Weber
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MWG Biotech AG
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MWG Biotech AG
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Priority to EP01112179A priority Critical patent/EP1260592A1/fr
Priority to US10/139,595 priority patent/US20030119014A1/en
Publication of EP1260592A1 publication Critical patent/EP1260592A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the invention relates to a biochip.
  • RNA Ribonucleic acids
  • sample may be an amplification step of the starting material over PCR (Polymerase Chain Reaction) or Invitrotranskription be inserted. It This is followed by data analysis, which is dependent on microarray hybridizations Of the integration density is often the most time-consuming step.
  • PCR Polymerase Chain Reaction
  • Invitrotranskription be inserted. It This is followed by data analysis, which is dependent on microarray hybridizations Of the integration density is often the most time-consuming step.
  • biochip technologies have the advantage of having the procedure of sample preparation over integrate the hybridization and detection up to the data analysis. By the farthest proceeding parallelization of analysis steps, the sample throughput can be increased (Medical Genetics No.1 March 1999 Volume 11 p.1-32).
  • An oligonucleotide chip consists of a carrier and the one for the respective experiment necessary, fixed on it molecules.
  • these molecules either in situ using photolithographic techniques using physical Masks synthesized on the matrix, or printed by various methods, such as the contact method using capillary needles or the Non-contact method based on piezoelectronic inkjet nozzles.
  • the production of imprinted DNA micro-arrays is divided into the activation or the Coating the solid chip matrix to the biomolecules via a suitable coupling chemistry be fixed.
  • Basic patent applications for DNA chips are z. EP 0 619 321 A, EP 0 373 203 A, EP 0 476 014 A and EP 0 386 229 A.
  • nucleic acid analysis various fluorescent dyes used as labeling agents.
  • Other detection options arise through the use of radioactive labels, chemiluminescent or otherwise Markings, by measuring the basis of these markings respectively detectable signals.
  • detection possibilities by means of impedance measurements are also possible or other physical measuring methods. It can in principle also a detection with the MALDI-TOF analysis (Mass Absorption Laser Desorption ionization-time of flight spectrometry) based on mass spectrometry, in which can be dispensed with labeling additives.
  • MALDI-TOF analysis Mass Absorption Laser Desorption ionization-time of flight spectrometry
  • RNA nucleic acid is from bases such as adenine (A), thymine (T), cytosine (C), guanine (G), uracil (U) or inosine (I). They are able to hybridize to double strands. Where A becomes T, C linked to G via hydrogen bonds. So-called base pairs form, e.g. AT or CG. In addition, there may be non-complementary base pairs z. B. AG, GU.
  • bases such as adenine (A), thymine (T), cytosine (C), guanine (G), uracil (U) or inosine (I). They are able to hybridize to double strands. Where A becomes T, C linked to G via hydrogen bonds. So-called base pairs form, e.g. AT or CG. In addition, there may be non-complementary base pairs z. B. AG, GU.
  • nucleic acids finds application in the detection of certain Nucleic acid sequences from sample material that must be prepared beforehand. There become nucleotide sequences complementary to the desired nucleic acid sequence provided that hybridize with the sequence sought. The formation of such Double strand must then be detected by suitable methods.
  • a such nucleotide sequence attached to the surface of a carrier may be by PCR techniques selectively amplified DNA or cDNA sections exist, which are specific to a particular transcript of an organism.
  • Oiigonucleotide chips exist with in situ synthesized oligonucleotides. Such Procedure is very expensive.
  • the oligonucleotides are built up step by step, wherein by means of templates one nucleotide after the other to the resulting An oligonucleotide is added.
  • oligonucleotides can also be synthesized ex situ on a support mash and attach covalently or otherwise to this. So made Biochips do not have the above-mentioned disadvantages and are for individual Applications available in stores.
  • An ex situ or ex vivo synthesis is emerging by the possibility of quality control, e.g. using MALDI-TOF (matrix assisted Laser desorption ionization time-of-flight mass spectrometry).
  • MALDI-TOF matrix assisted Laser desorption ionization time-of-flight mass spectrometry
  • nucleic acid sample to be examined is or the nucleic acid sequence complementary to oligonucleotide or Nucleic acid sequence immobilized on a solid phase.
  • the product can be washed.
  • the proof is i.d.R. over at the Hybridization or later incorporated radioisotopes, stains or other labeling agents, ultimately the subsequent detection of a signal on physical Pathways (C.R. Cantor, C.L. Smith, Genomics, John Wiley and Sons, New York 1999, p. 67).
  • Chips are executed in parallel, what the possible sample throughput per Time unit increased.
  • biochips The analysis of biological sample material by reaction with biochips leads to the chip surface immobilized molecules, their topology and quantities the biochip statements about the amount of a particular nucleic acid sequence, one Gene or a gene product in this sample material. Let it out biological information e.g. about gene expression and mutations in one Derive genome. These have a potential, accelerating benefit in the Development of new drugs, in the diagnosis and other areas of biomedical and biochemical research (M. Schena, R.W. Davis, Genes, genomes and chips in DNA microarrays, ed. M. Schena, Oxford University Press 1999).
  • Melting temperature is the temperature at which a potential hybrid is equal Parts double-stranded and single-stranded present. It depends on the type and composition the base pairs, from which a double strand composed and the nature and composition of the medium in which the duplex is present.
  • hybridization that is, the degree of hybrids, which is not mismatched in terms of non-complementary base pairs, is closely related to the stability of the formed hybrids together. Adjacent complementary base pairs afford depending on their respective nature and composition contributes to overall stability a hybrid. Is within a hybrid a mismatch in the sense of a non-complementary base pair, is missing compared to a faultless formed hybrid the contribution of a base pair as well as two cooperative interactions of two adjacent bound base pairs to the enthalpy of formation of those malformed hybrids. This is reflected in the lower melting temperature of the malformed Hybrid to the hybrids without mismatch. Thus, e.g.
  • the specificity of a hybridization depends on the length of the incoming into the hybrid Base fragments off. The more adjacent base pairs hybridize, the more faster is the actual hybridization. On the other hand, a faulty connection slows down the formation of a hybrid. This difference in speed increases with the Number of hybrid-forming base pairs. 10% mismatch in a given Hybrid slow down the reaction rate i.d.R. by half (R.J. Britten, E. H. Davidson, Hybridization strategy in Nucleic acid hybridization: a practical approach, Ed. B.D. James, S.J. Higgins, IRL Press 1985).
  • hybridization experiments are, for example, the detection of Mycoplasma pneumoniae (EP 0 173 920 A2), the detection of the protein human telomerase reverse transcriptase (hTRT) (EP 0 841 396 A1), and the detection of specific polymorphisms (eg EP 0812 922 A2). ,
  • Another important application of biochips is the analysis of gene expression patterns.
  • Gene expression patterns allow a statement about the expression certain genes of an organism depending on parameters, depending on Experiment be varied. It is desirable for this purpose in principle, the Degree of expression of as many, different genes at the same time on a biochip to be able to analyze, because thereby the time expenditure for the investigation of the Gene expression of as many genes of a genome can be minimized.
  • Qualitative statements about the degree of expression of different genes of a genome can be taken with high certainty, because statements about the Expression levels of many different genes of a genome using an analysis experiment under identical conditions as possible. in the In contrast to this would be statement about the expression of the same genes of a genome, which one would meet with the help of analysis experiments, the different ones Conditions may be less meaningful. That would be the case if you analyze the same number of gene expressions on different biochips would or if you have the same number of gene expressions with different trained probes, or methods would determine.
  • hybridizations are in addition to complementarity the base sequences that make up the hybrid, the length of the hybrid, their individual composition and their melting temperature.
  • a probe sequence on a biochip is specific to the Expression of a gene from the total amount of expressed genes of an organism detected.
  • ORFs open reading frames
  • An ORF is a region within a DNA molecule that, because of a particular initial sequence and sequence, is believed to be naturally expressible in a natural manner.
  • Escherichia coli An organism whose genome is known is Escherichia coli, a bacterium of the Enterobacteriaceae family. The presence of Escherichia coli and other bacteria in the intestinal flora is necessary for the physiological functions of the digestive system. In addition to the non-pathogenic Escherichia coli strains, there are also Escherichia coli strains which are infectious for humans. Escherichia coli has long been used as a model organism for the study of basic cellular and molecular processes, as well as for the analysis of reactions by which bakeries react to environmental influences, physiological changes or differentiation factors. In addition, populations of Escherichia coli are of great importance for the clinical production of amino acids and complex enzymes and proteins such as insulin, as well as an important source for the production of K and B complex vitamins.
  • the Escherichia coli K12 strain is established as a widely used model organism used as a reference strain for Gram negative bacteria. In order to be able to study the behavior of the model organism under different conditions (nutrient medium, temperature, etc.) and / or compare other organisms with it, it should be possible to study the expression of as many genes of an organism as possible.
  • the object of the invention is therefore to provide a device for comprehensive detection of the genome of Escherichia coli K12 available.
  • biochip according to claim 1, in which probes are provided on a support, which are suitable for the specific detection of genes or the activity of genes of Escherichia coli K12.
  • the probe sequences are nucleotide sequences, and at least one of the probes has probe sequences that are identical to or complementary to a portion of an open-reading frame of Escherichia coli K12.
  • the probe sequences are based on an inventive Biochip formed from synthetic oligonucleotides.
  • Such a trained Biochip has the advantage that the length of the probes according to certain criteria can be varied precisely, that a high purity and therefore selectivity and reactivity of the probes as well as reproducibility of with such a biochip carried out particularly well can be guaranteed.
  • the biochip has 4,289 specific probes for 4,289 different genes of the bacterium Escherichia coli K12.
  • Such an embodiment has the advantage that a complete set of genes of the genome of the model organism Escherichia coli K12 is provided, and thus a single device is used to analyze the expression of completely different genes at the same time.
  • a further preferred embodiment of a biochip according to the invention is a biochip which, in addition to probes for genes of the bacterium Escherichia coli K12, also has probes for mutants of Escherichia coli K12 and / or for other Escherichia coli strains.
  • biochips have the advantage that they can be used to compare gene expression in the model organism Escherichia coli K12 and in the corresponding mutants or other Escherichia coli strains in one and the same hybridization experiment.
  • a biochip in addition to qualitative and quantitative statements about the respective gene expression, it will also be possible to make such statements about the population density of the measured Escherichia coli strains or mutants.
  • Such a biochip can also be used for diagnostic procedures, for example, to determine which Escherichia coli strains are present in the intestinal flora of a patient.
  • a further preferred embodiment of a biochip according to the invention is a biochip which, in addition to probes for genes of the bacterium Escherichia coli K12, also contains probes for bacteria other than Escherichia coli K12.
  • Such a biochip has the advantage that it can be used for diagnostic purposes, in addition to the presence of the Escherichia coli K12 strain in a sample, the presence of other bacteria can be detected.
  • a selection procedure ensures that the probe sequences hybridize specifically with sample material that can be assigned to a specific gene or ORF of the species Escherichia coli K12.
  • the probe sequences are identical to or complementary to sequence sections from the genome of Escherichia coli K12.
  • a biochip according to the invention makes it possible to analyze gene expressions of Escherichia coli K12. It is demonstrated which genes of Escherichia coli K12 have been expressed to what extent.
  • Gen dar A sequence portion from the genome of an organism being expressed is set Gen dar. It is usually an ORF 1 (Open Reading Frame). When an ORF 1 is expressed in one step, this gene or ORF becomes 1 generates a certain amount of mRNA 2, this set of different parameters is dependent (Fig. 1).
  • ORF 1 Open Reading Frame
  • the generated mRNA 2 prepared by the generated mRNA 2 reverse transcribed into cDNA 3 becomes, whereby e.g. a fluorescent cy-3 or cy-5 marker incorporated into the cDNA 3 becomes (Fig. 1).
  • the cDNA 3 with a biochip according to the invention in combination placed on the nucleotide sequences attached to the cDNA molecules 3 can hybridize.
  • Such specific sequences can be identified from biological material amplify by PCR and apply to a biochip.
  • a preferred embodiment of the invention is one in which synthetic Oligonucleotides can be used for the preparation of the probes of a biochip.
  • oligonucleotide sequences that are used are in a through Computer calculations supported selection selected.
  • An ORF 1 that is n bases long may contain a sequence segment 5 that is m bases is long.
  • a n-long ORF 1 there are n-m different sequence sections 5 with a length of m bases. Accordingly, n-m can be different Identify oligonucleotide sequences 4 corresponding to n-m sequence segments 5 an ORF 1 are complementary and have a length of m bases.
  • nm oligonucleotide sequences 4 are identified which are complementary to possible sequence segments 5 of an ORF 1 of Escherichia coli ( Figure 2).
  • step S2 If the result is no, the process continues with step S2.
  • step S3 the oligonucleotide sequence 4 i is checked as to whether the GC content (GC) is in a predetermined range (between GC min and GC max ). For example, this range can be between 40 and 60%.
  • step S9 is entered, in which the oligonucleotide 4 i is discarded. This is followed by step S10, which terminates the test procedure for the oligonucleotide.
  • step S5 the oligonucleotide 4 i is checked to see if the calculated melting temperature tm is in a predetermined range.
  • step S9 is entered, in which the oligonucleotide 4 i is discarded. This is followed by step S10, with which the test procedure for the oligonucleotide 4 i is terminated.
  • step S6 is entered, in which it is checked whether the oligonucleotide 4 i can form secondary structures which make hybridization of the oligonucleotide 4 i with a complementary sequence segment 5 i difficult or unlikely. If this is the case, a branch is made to the steps S9, S10, with which the oligonucleotide 4; is discarded.
  • step S7 in which it is checked whether the oligonucleotide 4 i can form dimers which make it difficult or unlikely to hybridize the oligonucleotide 4 i with a sequence section 5 i complementary thereto. If this is the case, a branch is made to steps S9, S10, whereby the oligonucleotide 4 i is discarded.
  • step S8 it is checked in step S8 whether a cross-hybridization between the oligonucleotide 4 i and a sequence segment 5 j from a different ORF 1 than that on whose sequence segment 5 i the oligonucleotide 4 i was originally identified is possible. If the answer is yes, the system branches to steps S9 and S10.
  • step S12 from the set of oligonucleotides 4 i ⁇
  • 3 oligonucleotides are selected so that they hybridize furthest in the 3 'region of the ORF 1 and overlap each other a maximum percentage of a predetermined percentage.
  • sequence sections 5 i instead of the oligonucleotide sequences 4 i .
  • This may be the case if, instead of cDNA 3, it would like to detect mRNA 2 with an oligonucleotide chip according to the invention.
  • the steps S1 to S12 can therefore be carried out analogously for the selection of sequence sections 5 i .
  • FIG. 1 A section of a biochip according to the invention is shown in FIG.
  • the probe molecules 10 of a probe point 9 form a probe 11.
  • Each probe molecule 10 is composed of a linker 12, which is a covalent bond of the probe molecule to the support surface 8, a spacer 13, and a probe sequence 14th
  • a 5'-amino-modified oligonucleotide at the 5'-end of the spacer 13 is a linker 12.
  • the spacer 13 makes it possible to maintain a distance between the support surface 8 and the probe sequence 14.
  • a suitable spacer is, for example, a C 6 oligonucleotide which is composed of six dCTP monomers.
  • oligonucleotides which, between the amino-modification representing the linker 12 and the probe sequence 14, have a C 6 -pacer 13 composed of six dCTP-monomers.
  • the oligonucleotides are dissolved to a concentration of 100 .mu.M in 50 .mu.M Na borate (pH 8.5) and 250 mM NaCl, on CSS silylated slides from CEL Associates, Houston USA, using an Affymetrix 417 arrayer spotted, rehydrated in a humidity chamber and dried at room temperature. Subsequently, the formation of the covalent bond between linker and support surface is completed by a reduction with NaBH 4 .
  • An oligonucleotide sequence 4 selected by the method explained above may be a probe sequence.
  • the probe sequence is at the 5 'end bonded to the spacer 13.
  • the probe sequences 14 of a probe 11 differ from the probe sequences 14 of other probes 11. Within one probe point 9, only identical probes 11 are provided. However, it is within the scope of the invention also possible, different probe sequences 14 within a probe 11 provided.
  • an oligonucleotide sequence 4 is determined for each of 4,289 ORFs in the genome of Escherichia coli K12, which is used as probe sequence 14.
  • the determined probe sequences 14 are complementary to sequence sections 5 from the genome of Escherichia coli K12, which are given in the attached sequence listing.
  • sequence regions ⁇ 210> are indicated for 4,289 different ORFs 1, whereby the sequence regions ⁇ 210> specified for an ORF 1 ( ⁇ 220>) are in each case of all other specified sequence regions ⁇ 210> of the remaining ones ORFs 1 ( ⁇ 220>) of the genome of Escherichia coli K12 differ.
  • sequence regions ⁇ 210> belonging to an ORF 1 are listed immediately after one another in the sequence listing.
  • a portion of a predetermined length of one of the sequence areas ⁇ 210> is for the respective ORF 1 specific.
  • the minimum length is 20 bases.
  • probe sequences sections are preferred which are the 20 bases of the core regions according to the key number ⁇ 313> of the respective sequence areas ⁇ 210>. These core regions or their complements form particularly preferred oligonucleotide sequences 4 or probe sequences 14 ( Figure 4) because they are specific.
  • probe sequences are also specific, if the number of bases is at least 40.
  • sequence regions are below ⁇ 210> by SB (S FREQUENCY b rea) followed by her in the sequence listing by the code ⁇ 210> specified sequence identity number and the core areas of ⁇ 313> by kilobytes (K s b rea) followed by the sequence identity number specified sequence range specified.
  • each oligonucleotide sequence 4 is complementary to a sequence section 5 of the respective ORF 1 ( ⁇ 220>) which is 9 bases before respective core area KB begins and ends 11 bases after that subsection.
  • 4,416 probes On a support a total of 4,416 probes are applied. 4,289 of these probes have 40-base probe sequences 14 corresponding to the 4,289 oligonucleotide sequences 4 described above. The remaining 127 probes are control probes. Of these, 79 are replicates of complements of portions of the genome of Escherichia coli K12 that always hybridize (positive control), the remaining 48 probes those that have probe sequences that are incapable of hybridizing to a transcription product from the expressed genome of Escherichia coli K12 ( Arabidopsis negative controls).
  • Each of the 4,289 probes hybridizes to a different cDNA molecule 3, the from each of an ORF 1 ( ⁇ 220>) expressed mRNA 2 reverse transcribed becomes.
  • the probes are deposited on the support on an area of 2 x 2 cm 2 as described above.
  • probe sequences 14 than those in the embodiment specified oligonucleotide sequences 4 applied to the biochip as long as they are part of a sequence range specified for the respective ORFs 1 ( ⁇ 220>) ⁇ 210> are.
  • An advantageous embodiment of a biochip according to the invention is a biochip on which probe sequences for the detection of sample material (eg cDNA 3 or mRNA 2) from Escherichia coli K12 cultures are provided and on which additional probes for the detection of mutations of Escherichia coli K12 are provided within this sample material are.
  • sample material eg cDNA 3 or mRNA 2
  • An advantageous embodiment of a biochip according to the invention is a biochip on which probe sequences for the detection of sample material (eg cDNA 3 or mRNA 2) from Escherichia coli K12 cultures are provided and on which additional probes for detecting other bacterial strains within the sample material are provided.
  • sample material eg cDNA 3 or mRNA 2
  • additional probes for detecting other bacterial strains within the sample material are provided.
  • free choice allows the length of probe sequences 14, the preferred Melting temperature of the calculated hybrid 6 and that for the respective calculations used a more accurate matching.
  • oligonucleotide sequences with different Provide lengths in individual probe points may also be appropriate.
  • Two nutrient solutions A and B are applied with Escherichia coli K12, where nutrient solution A is exposed to conditions other than nutrient solution B.
  • the different conditions are the parameters whose effect on gene expression is investigated.
  • Such parameters may be, for example, temperature, concentration and / or special ingredients of the nutrient solutions (FIG. 5a)).
  • a first step the respective mRNA 2 of the nutrient solutions A and B in cDNA 3 transcribed, in which a cDNA transcript cy-5-labeled bases incorporated and in the other cDNA transcript cy-3-labeled bases are incorporated (Fig. 5b)).
  • the difference between these two measured values yields a corrected value for the fluorescence intensity of the cDNA molecule 3. which has formed a hybrid 16 in the measured probe point 8 and is provided with the corresponding marker.
  • This corrected intensity value is proportional to the amount of ORF 1 expressed by Escherichia coli K12 in one of the two nutrient media for which the probe 11 is specific.
  • the totality of the intensity values determined in one channel corresponds to a gene expression pattern which provides information about the expressions of those ORFs 1 from Escherichia coli K12 for which specific probes 9 are provided on the biochip.
  • the gene expression pattern depends on the conditions to which the respective sample Escherichia coli K12 is exposed before the mRNA 2 is transcribed. It is assumed that the majority of the genes, or ORFs 1 of an organism, that is about 85%, is the same expression under different conditions. If one thus determined several expression patterns of the complete genome of an organism, two different expression patterns would each have only a part of genes which are present in different concentrations.
  • the evaluation is done by dividing the differences between the multiplied by the factor determined intensity values of the total low-intensity channel and the determined intensity values of the other channel, namely for every single probe point.
  • the differences between the normalized, determined intensity values allow a qualitative statement about the different expression of a gene among different To make conditions.
  • the determined intensity values also allow a statement about how much more a particular gene under conditions A was expressed as under conditions B.
  • an inventive Biochip will be made a quantitative statement by means of scaling.

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Cited By (7)

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WO2005108578A1 (fr) * 2004-05-07 2005-11-17 Warnex Research Inc. Polynucleotides pour la detection d'escherichia coli
US7329742B2 (en) * 2003-09-04 2008-02-12 The Regents Of The University Of California Aptamers and methods for their in vitro selection and uses thereof
WO2010132832A1 (fr) * 2009-05-15 2010-11-18 Life Technologies Corporation Dosage specifique de e. coli o157:h7
WO2011140599A1 (fr) * 2010-05-11 2011-11-17 Monash University Réseaux de composés chimiques utiles pour la détection de peptides bactériens
US20180371462A1 (en) * 2014-02-18 2018-12-27 Somalogic, Inc. Compositions and Methods for Detecting Microorganisms
US20210102249A1 (en) * 2013-07-26 2021-04-08 Life Technologies Corporation Control nucleic acid sequences for use in sequencing-by-synthesis and methods for designing the same

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JP3788513B2 (ja) * 2003-09-11 2006-06-21 セイコーエプソン株式会社 固相基板上への分子の固定化方法およびそれを用いるバイオセンサの製造方法
KR101803472B1 (ko) * 2016-08-11 2017-12-28 한국과학기술원 유전체 유래 인공 ncRNA 발현 라이브러리 및 이의 제조방법

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WO2005108578A1 (fr) * 2004-05-07 2005-11-17 Warnex Research Inc. Polynucleotides pour la detection d'escherichia coli
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WO2010132832A1 (fr) * 2009-05-15 2010-11-18 Life Technologies Corporation Dosage specifique de e. coli o157:h7
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